Project Summary Cannabinoids are bioactive natural products with many current and potential theoretical therapeutic uses that are generally extracted from natural plant sources. While the cannabinoids tetrahydrocannabinolic acid (THCA), and cannabidiolic acid (CBDA) are the highest abundance and therefore the most well studied, there are many other low abundant (rare) cannabinoids that are also made in plants (e.g. the ?varins? cannabidivarinic acid, CBDVA, and tetrahydrocannabivarinic acid, THCVA). Plant production of cannabinoids, particularly rare cannabinoids, is problematic because of crop variability, purification challenges and environmental concerns. Consequently, there is considerable interest in producing both common and rare cannabinoids by metabolic engineering of microbes. Microbial production of cannabinoids also faces daunting challenges, however, and published titers so far are several orders of magnitude below cost competitive levels (8 mg/L). Invizyne Technologies is developing an alternative, cell-free method to produce common and rare cannabinoids (and other natural products) using enzymatic transformations. Our primary focus is production of the central cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA), because a variety of important cannabinoids can be produced from CBGA/CBGVA in single enzymatic steps. Moreover, CBGA itself is bioactive and shows promise for treatment of glaucoma, inflammatory bowel disease, and Huntington?s disease. A key barrier to cell-based and cell-free production of cannabinoids has been reliance on the native enzyme that makes CBGA/CBGVA, Geranyl:Olivetolate Transferase (GOT), which is a membrane protein. In a major development, we were able to design a highly active, specific and water soluble GOT enzyme. With this GOT enzyme in hand, we have designed a 7 enzyme system for the production of CBGA/CBGVA, that we call SimplePath. Not only is a 7 enzyme system tractable for commercial development, but initial tests provide CBGA titers well over 1 g/L, already far exceeding cell-based methods by several orders of magnitude. Our goal in this Phase I application is to expand our SimplePath approach and make necessary improvements to lower costs and improve titers even further. At the end of Phase I we will perform techno-economic analysis on the optimized SimplePath system to guide commercialization efforts (and identify weak points) that will be addressed in a Phase II project. Phase I work is necessary for establishing a consistent, highly pure supply of cannabinoids to be used as therapeutics.